Peter Scheurich

Fluorescent photoaffinity labeling of F1 ATPase from Micrococcus luteus with 8-azido-1,N6-etheno-adenosine 5′-triphosphate

Abstract A fluorescent photoaffinity label—8-azido-1-N6-etheno-adenosine 5′-triphosphate (8-N3e ATP)—for ATP-binding proteins has been synthesized. The effectiveness of the label is demonstrated with F1ATPase from Micrococcus luteus. 8-N3e ATP is a substrate for the enzyme in the presence of bivalent cations. Ultraviolet irradiation of F1ATPase in the presence of the label and Mg2+ ions inhibits the enzyme irreversibly. The fluorescent label is bound preferentially to the β subunit of the enzyme. Labeling and inactivation are decreased by protection with ATP or ADP.

Photoaffinity labeling of the coupling factor 1 from the thermophilic bacterum PS3 by 8-azido ATP

To localize the nucleotide binding sites of the F1ATPase (TF1) from the thermophilic bacterium PS3 we have used 14C‐labeled 8‐azido ATP (8‐N3ATP) as photoaffmity label. 8‐N3ATP is hydrolyzed by the F,ATPase in the absence of ultraviolet light. Irradiation by ultraviolet light of the enzyme in the presence of 8‐N3ATP results in reduction of ATPase activity and in preferential nucleotide specific labeling of the α subunits (0.8–0.9 mol 8‐N3ATP/TF1,α:β = 4:1). Inactivation and labeling do not depend on the presence of Mg2+. Both effects decrease upon addition of various nucleotide di‐ or triphosphates.

Frequency-Analysis of Precursors of Cytotoxic T Lymphocytes in Radiation Chimeras: Enumeration of Antigenspecific CTL-P Restricted to Thymic MHC- and Bone Marrow-MHC-Determinants

The mechanisms controlling the acquisition of T cell restriction specificity and immunocompetence are, despite of numerous investigations, not well understood. From studies of the CTL-immune responsiveness in thymus- and bone marrow-grafted chimeric mice, it became apparent, that it is the thymus which is crucial not only for the maturation or T cells, but also for the specificity repertoire of the T cells (1,2). From these data it was suggested, that during intra-thymic maturation both mutational events and positive selection mechanisms influence the repertoire such that only T cells restricted to thymic epithelial cell MHC determinants mature and will be exported to the peripheral lymphoid organs (3,4). However, the demonstration of non-thymic MHC restricted CTL in both chimeric (5–7) as well as conventional mice (8–10) are incompatible with models proposing strictly thymus-dependent selection mechanisms. Allo-MHC restricted T cells were found not only within spleen cells, but also within thymocytes of both chimeric (9) and non-chimeric mice (10). Accordingly, both self- and allo-MHC restricted thymocytes mature to immunocompetent T cells.